PyroMark Q24 MDx

When using the PyroMark Q24 v2.0, PyroMark Q24 Advanced v3.0, and PyroMark ID 2.5 software, the Sequence to Analyze can be modified if an unexpected mutation is detected. Enter the Sequence to Analyze that fits the detected mutation and apply the change to re-analyze the data.

When using the PyroMark ID 1.0 and PyroMark MD 1.0 software, the Sequence to Analyze cannot be modified. If an unexpected mutation is detected, you will need to re-run the assay with the new Sequence to Analyze.

Cracked waste bottle cap, dirty or wet inline filter between the waste bottle and the vacuum pump, and more than 100-time usage of the vacuum prep probes are the most common causes of slow suction.

Check the integrity of the waste bottle cap. Replace the dirty or wet inline filter. Each system comes with two inline filters. You can order extra inline filters from Millipore with catalog number SLFG05010. Replace the probes after 100 times of usage.

This instruction is intended to retrieve log file for PyroMark Q96 ID, PyroMark Q96 MD/MDA, PSQ MA, and PSQ HS/HSA instruments

1. Power on both the operator’s computer and the PyroMark instrument. Wait until the Information LED on the instrument’s front panel is blinking.

2. On the operator’s computer, click the Start button and click Run…. A new window will open.

3. In the new window, type the string \\192.168.255.201\C$ and click OK. A login window will open.

4. Log in with user name Administrator and leave the password field blank. Some instruments may have the user name Pyroservice and password floang.

Note: User names and passwords are case sensitive.

5. In the new window, sequentially open the “Pyrosequencing”, the “Instrument”, and then the “Log” folders.

6. Copy the “PSQXXXLog.txt” file to the Operator’s computer and send it to QIAGEN Technical Service for further assistance.

Blocked reagent cartridge/tips, reagent issue, and incorrect pipetting are the common causes of the substrate peak being absent. Test cartridge/tips before adding reagent. Ensure reagent is pipetted to the correct positions and is collected at the bottom of the cartridge/tips.          

If the substrate peak is present, loss of template during the vacuum prep steps, no or incorrect sequencing primer, and camera failure are the common causes. Ensure no residual vacuum pressure before releasing the streptavidin beads and that the correct sequencing primer is used. Perform camera function test, see FAQ ID 9046.

If the issue persists, please send the run files (see FAQ ID 9048) to QIAGEN Technical Service for further assistance.

The camera function test is intended for the PyroMark Q96 ID, PyroMark Q96 MD/MDA, PSQ MA, and PSQ HS/HSA instruments.

1. Setup a mock run without using PyroMark reagent and other consumables.

2. When nucleotide dispensation starts, open both instrument lids so that the 96-well block is exposed to ambient light.

3. Check the detected signal level on the Y-axis on the operator’s computer and report the value to QIAGEN Technical Service.

Note: The inner lid of the instrument moves around during nucleotide dispensation. The lid can still be lifted while it is moving.

For PyroMark Q24 and PyroMark 24 Advanced instruments, please contact QIAGEN Technical Service for the self-test software and instruction.

If you need to install the PyroMark Q24 software on a new computer replacing your old one, or after you reinstall or upgrade the operating system, you can reinstall the PyroMark Q24 software without purchasing a new license.

Insufficient amount of PCR template, loss of streptavidin bead during vacuum prep steps, incorrect instrument method, and incorrect reagent are the common causes of low peak signals.

Check the PCR product with QIAxcel instrument or agarose gel electrophoresis and determine the appropriate amount of PCR product to be used. Perform vacuum prep function test. Determine the vacuum prep station functionality using the PyroMark Control Oligo with and without the vacuum prep steps. Ensure the correct instrument method is used. Refer to Managing PyroMark Instrument Methods. Ensure the correct reagent is used for the specific instrument model.

PyroMark instruments dispense the correct volume of enzyme mix, substrate mix, and nucleotides using variable pulse times and dispensing pressures. The settings for pulse times and dispensing pressure are specific for each lot of PyroMark dispensing tips and cartridges and need to be updated in the PyroMark software being used to operate the instrument.

Dispensing pressures and pulse time settings must be checked and, if necessary, updated every time a cartridge from a new lot number is used. Check the details at Managing PyroMark Instrument Methods.

Clogged cartridge/tips, and excessive PCR product are the most common causes. Test cartridge/tips before adding reagent. Avoid using filtered pipette tips because small particles from the filter can clog the cartridge/tips. Use appropriate PCR product for optimal single peak heights, also see FAQ ID 9058.

If the issue persists, please send the run files (see FAQ ID 9048) to QIAGEN Technical Service for further assistance.

Please see the Pyrosequencing Technology and Platform Overview.

Fill a PCR plate with 80– 100 µl of high-purity water. Apply vacuum pressure to the vacuum prep tool. Lower the vacuum prep tool to the PCR plate and wait for 10 seconds. If the wells are not empty in 10 seconds, the vacuum system needs to be checked. Check the integrity of the waste bottle cap. Replace the inline filter if dirty or wet. Replace the vacuum probes after they are used 100 times.

Sodium hydroxide carry-over, too much template, and incorrectly stored reagent are the most common causes of wide peaks. Ensure correct volume of denaturation buffer is used on the vacuum prep station. Use 5–20 µl of PCR product depending on instrument models. Store reagent as described in the handbook.

If the issue persists, please send the run files (see Instruction for run file export) to QIAGEN Technical Service for further assistance.

For PyroMark Q24, PyroMark Q24 Advanced, PyroMark Q96 ID v2.5, and PyroMark CpG software, data are file-based. The original run files can be attached to email and sent to QIAGEN Technical Service.

For PyroMark Q96 ID 1.0, PyroMark Q96 MD/MDA 1.0, PSQ MA, and PSQ HS/HSA software, data are stored in an Oracle database and need exporting (See Instruction for run file export). Exported data can be attached to email and sent to QIAGEN Technical Service.

The log files from the PyroMark Q96 ID, PyroMark Q96 MD/MDA, PSQ MA, and PSQ HS/HSA instruments provide valuable information. See Instruction for log file retrieval) for log file retrieval instruction

Please send the log file along with the original run file to QIAGEN Technical Service.

A single peak height is the signal produced from a non-variable single nucleotide.  The single peak height is an important criterion for trouble-shooting.

The optimal single peak height is 30 RLU (minimum 20 RLU) (Relative Light Unit) for PyroMark Q24 and PyroMark Q24 Advanced, 20 RLU (minimum 10 RLU) for PyroMark Q96 ID, and 100 RLU (minimum 50 RLU) for PyroMark Q96 MD instrument, respectively.

The PyroMark Q96 ID 1.0, PyroMark Q96 MD/MDA 1.0, PSQ MA, and PSQ HS/HSA software requires a functional Oracle database, which cannot be installed on an office computer. You can install the PyroMark software on an office computer and access the Oracle database on the PyroMark operator’s computer via network. The operator’s computer needs to be powered on while you are accessing the Oracle database remotely. Your IT department is responsible for the network setup.

1. When the instrument is not processing, insert a USB stick into the USB port on the instrument.

2. Use the Up and Down buttons to select “Administration” in the menu and press OK.

3. Select “Copy Log Files” and press OK. When the instrument confirms that the log files have been saved to the USB stick, press Close and remove the USB stick.

For other PyroMark instruments, see FAQ ID -9047

The instruments can continue processing samples if there is no USB stick inserted in the USB port. The data are stored in an internal storage memory card. Follow these steps to retrieve data from the internal storage memory.

1. When the instrument is not processing, insert a USB stick into the USB port on the instrument.

2. Use the Up and Down buttons to select “Administration” in the menu and press OK.

3. Select “Copy Unsaved Runs” and press OK.

4. Use the Up and Down buttons to select the run file for retrieval and press Select.

5. When the instrument confirms that the run file has been saved to the USB stick, press Close and remove the USB stick.